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Blood Clotting Factor VIII
Factor VIII is a protein which is essential in blood clotting. It is synthesized as a 300 kDa pre-protein. It is secreted as a heterodimer following at least two cleavages at the B-domain. In circulation it non-covalently associates with von Willenbrand factor in its inactive form. 1 These proteins dissociate from one another when an injury occurs and factor VIII associates with factor IX and helps to mediate the coagulation cascade resulting in a blood clot. Individuals with hemophilia a have a defect in the F8 gene, resulting in clotting deficiencies. The defective factor VIII gene is responsible for approximately 80% of cases of hemophilia. It is also a heritable, sex-linked mutation located on the X chromosome. In the past, these patients would need to have blood transfusions to replenish factor VIII in their bloodstream. This came with additional risks of transmission of blood-borne pathogens, particularly hepatitis B and C and HIV. 2 Recombinant Factor VIII In 1984, a recombinant factor VIII was successfully cloned and expressed. It is a highly complex protein with multiple glycosylation sites and, as such, needs to be expressed using mammalian cell lines. The original cloned and expressed recombinant factor VIII was made as an expression plasmid with the factor VIII coding region in between an SV40 early promoter/adenovirus-2 major late promoter and hepatitis B surface antigen gene polyadenylation sequence. The plasmid also included a murine DHFR gene under the conrol of an SV40 early promoter for selection. The plasmid was transfected into baby hamster kidney cells using calcium-phosphate coprecipitation. The protein was purified using immunoaffinity chromatography. 3 As of 2001, several commercially suppliers of rFVIII (recombinant factor VIII) which can be split into first generation and second generation products have been described. The first generation products, Kogenate/Helixate Clinical and Recombinate, are full-length molecules grown in human serum albumin. Kogenate uses baby hamster kidney cells for expression and is cloned using a plasmid vector with a tandem promoter and a dihydrofolate reductase gene. Purification uses multiple chromatography methods, beginning with anion exchange chromatograpy and virus inactivation then immunoadsorption chromatography, followed by gel filtration for size exclusion. The immunoadsorption chromatography step is repeated followed by a final anion exchange step. Recombinate uses chinese hamster ovary cells and a plasmid vector that also has a DHFR gene. Methotrexate is used for amplification and human Von Willenbrand factor is also added to aid in expression. Purification of the protein is done in a 3-chromotography step process. This begins with immunoaffinity chromatography followed by two ion exchange chromatography steps. Second generation products, not grown in the presence of human serum albumin but alternatively in the presence of sucrose are Refacto and Kogenate Bayer/Kogenase FS. Similar to Recombinate, Refacto uses CHO cells and contains a DHFR gene and uses methotrexate as an amplifier. Unlike the previous proteins, Refacto is not a complete protein but lacks a B-region and instead the N and C terminal of the B region are fused together with a peptide linker. This also aids in stability. Purification is a 5-step chromatography process with a virus inactivation step. The newest product, Kogenate Bayer/Kogenate FS, is a full protein made in serum free conditions. The protein is made in BHK cells, similar to Kogenate. A six-step chromatography step is used for purification and a detergent based viral inactivation step is used. The purification process utilizes anion-exchange, immunoaffinity, immobilized metal affinity, gel filtration and cation-exchange chromatography. 4 Studies have shown recombinant factor VIII as a safe and effective treatment for hemophilia a. The New England Journal of Medicine published a comprehensive study in 1993 detailing the high level of tolerance of Factor VIII with extremely low levels of adverse reactions. They also showed a high level of efficacy of the treatment. In 1992 Baxter in conjunction with Genetics Institute introducted the first genetically engineered Factor VIII concentrate plasma. Recombinant factor VIII carries less risk and is less immunogenic than plasma-derived factor VIII and increases the quality of life and lifespan of those with hemophilia a. 5,6 The Future of Recombinant Factor VIII Short half-life of recombinant factor VIII and the production of antibodies against the introduced therapy are two problems currently being faced by patients with hemophilia a. PEGylation of recombinant factor VIII is one method currently being researched to potentially increase half-life and decrease immunogenicity of recombinant factor VIII. This requires the addition of mono-methoxy PEG to outside amino acid residues of the protein, particularly lysine residues. Polysialic acid addition is another protein modification which could potentially stabilize and increase efficacy of recombinant factor VIII treatment. Factor VIII could also be fused to another protein with a longer half-life to increase treatment efficiency. [5 References 1. Fang, Hong et. al. The Protein Structure and Effect of Factor VIII. Thrombosis Research. Volume 119, Issue 1 2007. pp.1-13. http://www.sciencedirect.com/science/article/pii/S0049384806000028 2. Genetics Home Reference. F8. May 2010. http://ghr.nlm.nih.gov/gene/F8 3. Wood, William, ''et. al. ''Expression of the Human Factor VIII from Recombinant DNA Clones. Nature. Volume 312, 22 November 1984. http://europepmc.org/abstract/MED/6438526 4. Schwartz, Richard ''et. al. ''Human Recombinant DNA-derived Antihemophilic Factor (Factor VIII) in Treatment of Hemophilia A. The New England Journal of Medicine. Dec. 27 1990. http://www.nejm.org/doi/pdf/10.1056/NEJM199012273232604 5. Lusher, Jeanne ''et. al. ''Recombinant Factor VIII for the Treatment of Previously Untreated Patients with Hemphilia A. The New England Journal of Medicine. Feb. 18 1993. Volume 328, No. 7. http://www.nejm.org/doi/pdf/10.1056/NEJM199302183280701